壳聚糖-明胶海绵与脐带间充质干细胞修复皮肤伤口的差异

doi:10.3969/j.issn.2095-4344.2016.34.019

摘要/Abstract

摘要：

文章快速阅读：

文题释义：
影响伤口愈合的全身因素：①年龄：青少年的组织再生能力强，愈合快。老年人则相反，组织再生能力差，愈合慢，与老年人血管硬化、血液供应减少有很大的关系；②营养：严重的蛋白质缺乏，尤其是含硫氨基酸缺乏时，肉芽组织及胶原形成不良，伤口愈合延缓。维生素中以维生素C对愈合最重要。
影响伤口愈合的局部因素：①感染与异物感染。许多化脓菌产生一些毒素和酶，能引起组织坏死，基质或胶原纤维溶解。这不仅加重局部组织损伤，也妨碍愈合。伤口感染时，渗出物很多，可增加局部伤口的张力，常使正在愈合的伤口或已缝合的伤口裂开，或者导致感染扩散加重损伤；②局部血液循坏。局部血液循环一方面保证组织再生所需的氧和营养，另一方面对坏死物质的吸收及控制局部感染也起重要作用；③完整的神经支配对组织再生有一定的作用。自主神经的损伤，使局部血液供应发生变化，对再生的影响更为明显。④电离辐射能破坏细胞，损伤小血管，抑制组织再生。因此能阻止瘢痕形成。
脐带间充质干细胞的鉴定标准：①在标准培养条件下呈贴壁生长；②表达CD105，CD73和CD90，不表达CD45，CD34，CD14或CD11b，CD79a或CD19和HLA2DR；③在体外至少能向成骨细胞、脂肪细胞和软骨细胞分化。

背景：脐带间充质干细胞有多向分化和自我复制能力，在一定条件下可以向骨细胞、软骨细胞、平滑肌细胞、神经细胞以及血管内皮细胞分化，有助于表皮再植和血管生成。
目的：比较脐带间充质干细胞与壳聚糖-明胶海绵在伤口愈合方面的作用。
方法：将30只雄性SD大鼠等分为模型组、壳聚糖-明胶海绵组和脐带间充质干细胞组。3组大鼠均在背部建立3 cm×3 cm全层皮肤缺损创面。造模后1 d起，模型组连续15 d皮肤伤口边缘注射PBS；壳聚糖-明胶海绵组连续15 d在皮肤伤口边缘注射壳聚糖凝胶；脐带间充质干细胞组在皮肤伤口边缘注射脐带间充质干细胞。
结果与结论：与模型组相比，壳聚糖-明胶海绵组和脐带间充质干细胞组大鼠创面愈合率较高，表皮厚度降低，胶原纤维面积升高；且脐带间充质干细胞组效果优于壳聚糖-明胶海绵组。说明壳聚糖-明胶海绵和脐带间充质干细胞均可有效修复皮肤损伤，且脐带间充质干细胞效果更佳。

ORCID: 0000-0002-2237-4969(方少霞)

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

关键词: 生物材料, 材料相容性, 脐带间充质干细胞, 壳聚糖-明胶海绵, 胶原纤维, 伤口愈合, 间充质干细胞

Abstract:

BACKGROUND: Umbilical cord mesenchymal stem cells possess multipotent differentiation and self-replication abilities, and can differentiate into osteoblasts, chondrocytes, smooth muscle cells and vascular endothelial cells, which contribute to reepithelialization and angiogenesis.
OBJECTIVE: To compare effects of umbilical cord mesenchymal stem cells and chitosan-gelatin sponge in skin wound healing.
METHODS: Thirty male Sprague-Dawley rats were equivalently randomized into model, chitosan-gelatin sponge and stem cell groups. 3×3 cm full-thickness skin defects were created on the rat dorsal area in each group, and 1 day later, model rats in each group were given intradermal injection of PBS, chitosan gel or umbilical cord mesenchymal stem cells, respectively, for successive 15 days.
RESULTS AND CONCLUSION: Compared with the model group, the wound healing rate was higher, the epidermal thickness was decreased, and the collagen fiber area was larger in the chitosan-gelatin sponge and stem cell groups. Furthermore, the healing effect in the chitosan-gelatin sponge group was superior to that in the stem cell group. To conclude, both chitosan-gelatin sponge and umbilical cord mesenchymal stem cells, especially umbilical cord mesenchymal stem cells, can effectively repair skin defects.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

Key words: Skin, Wound Healing, Tissue Engineering

中图分类号:

R318

方少霞，郭芬. 壳聚糖-明胶海绵与脐带间充质干细胞修复皮肤伤口的差异[J]. 中国组织工程研究, 2016, 20(34): 5136-5141.

Fang Shao-xia, Guo Fen. Chitosan-gelatin sponge versus umbilical cord mesenchymal stem cellsfor skin wound healing[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(34): 5136-5141.

图/表（结果） 3

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引言

脐带间充质干细胞是从脐带组织中分离出来的具有多向分化潜能的细胞^[1] ，目前已证实其在体外可以向骨细胞、软骨细胞、平滑肌细胞、神经细胞以及血管内皮细胞分化^[2-6] 。由于脐带来源丰富、容易获取、患者痛苦小，且无免疫排斥，因而脐带间充质干细胞被认为较其他成体干细胞具有更加广阔的应用前景。有研究显示脐带间充质干细胞比骨髓脐带间充质干细胞分泌更多的血管生成生长因子，如血管内皮生长因子、胰岛素样生长因子1、肝细胞生长因子等^[7-8] ，还可表达多种细胞因子和迁移相关基因^[9-10] 。研究显示，脐带间充质干细胞可以通过巨噬细胞刺激蛋白释放促进伤口愈合^[11-12] 。因而脐带间充质干细胞可能有助于表皮再植和血管生成^[13] 。另外，脐带间充质干细胞还可通过旁分泌增加血管生成，促进伤口愈合^[14] 。
壳聚糖是甲壳素经乙酰基反应后得到的可溶性物质，由于其具有良好的生物相溶性、生物降解性、成膜性、抗凝血性，能促进伤口愈合和防腐抗菌等^[15-16] 。壳聚糖可通过增强细胞免疫应答促进免疫调节^[17] ，还可诱导局部巨噬细胞增生及活性增强^[18-20] ，并促进肉芽组织形成，炎症细胞迁移及伤口早期愈合^[21-24] ，同时还可刺激伤口附近的细胞外基质胶原纤维的重塑^[25] 。明胶的主要成份是胶原、少量的蛋白多糖及某些有机成分^[9] ，能参与细胞的迁移、分化和增殖，使骨、腱、软骨和皮肤具有一定的机械强度，还能促进创伤愈合^{[9-10，15，26]} 。
作者拟通过观察脐带间充质干细胞及壳聚糖明胶对皮肤愈合面积及纤维生成面积，皮肤厚度的变化，为临床应用脐带间充质干细胞及壳聚糖明胶修复皮肤创面提供依据。

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

材料方法

1.1 设计随机对照动物实验。

1.2 时间及地点实验于2015年1至10月在华中科技大学生物医学研究所完成。

1.3 材料 SPF级雄性SD大鼠30只，体质量150- 200 g，购自北京大学医学部实验动物科学部。许可证号SYXK(京)2015-0039。大鼠饲养于温度18-22 ℃，相对湿度50%-60%，噪声< 85 dB，氨浓度20 ppm。实验经华中科技大学动物伦理委员会批准。

脐带取自足月剖宫产健康孕妇，孕妇HBV抗原、抗HCV抗体、抗HIV抗体、抗梅毒螺旋体抗体、支原体、抗巨细胞病毒抗体等检测均阴性。取得孕妇及家属同意，并签字留样，实验经华中科技大学附属同济医院伦理委员会批准。

壳聚糖购于天津灏洋生物制品科技有限责任公司。明胶由中国科学院遗传与发育生物学研究所戴建武研究组惠赠。

1.4 方法

1.4.1 脐带的前期准备无菌条件下采集脐带后送往实验室，如不能立即制备应将脐带置保存液内于 4 ℃冰箱存放，且不得超过6 h。

1.4.2 脐带间充质干细胞制备使用改良胶原酶消化法获得脐带间充质干细胞。取华尔通胶，剪碎成0.5- 1.5 mm³的组织块，加入1.0 g/L Ⅳ型胶原酶，于37 ℃恒温振荡培养箱振荡消化3 h后，用80目滤网过滤，将滤过的细胞悬液离心洗涤。用无血清培养液混匀细胞，以1.0×10⁵/cm²接种于75 cm²培养瓶中，置37 ℃、饱和湿度、体积分数5% CO₂培养箱中培养。细胞贴壁后，每隔3或4 d换液一次。融合至80%-90%传代培养，加入2.5 g/L胰酶(含EDTA)消化，按1∶2或1∶3传代至175 cm²培养瓶中^[27-29]。

1.4.3 壳聚糖-明胶海绵状伤口敷料的制备采用壳聚糖-明胶为原料配成溶液，加入适当的交联剂及其他助剂，成型冷冻，经低温下骤冷、固化，再脱水，形成1.0- 2.0 mm厚的具有独特膜孔结构海绵伤口敷料。消毒备用。

1.4.4 皮肤伤口动物模型的制备将30只SD雄性大鼠随机等分为模型组、壳聚糖-明胶海绵组及脐带间充质干细胞组。术前3 d剔除背部鼠毛并行戊巴比妥钠(40 mg/kg)腹腔注射麻醉后，在背部作3 cm×3 cm全层皮肤缺损创面。

1.4.5 干预造模后1 d起，模型组在大鼠皮肤伤口边缘注射0.5 mL PBS，连续15 d。壳聚糖-明胶海绵组在大鼠皮肤伤口边缘注射0.5 mL壳聚糖凝胶，1次/d，连续15 d。脐带间充质干细胞组在大鼠皮肤伤口边缘注射脐带间充质干细胞(1×10⁶)，每侧给予0.25×10⁶个细胞。

各组创面予相应处理后，异体皮覆盖^[28]，缝合固定，复苏后单笼饲养于SPF级动物房。术后观察大鼠体征、行为活动、尿粪性状及创面愈合情况，

1.4.6 创面愈合效果薄膜描记法获取未愈创面面积。创面愈合率=(原始创面总面积-未愈创面面积)/原始创面总面积×100%。

修复后10-15 d，硫喷妥钠麻醉，取伤口周围全厚皮肤，通过测量伤口周围皮肤厚度，评价表皮厚度的变化，用IPP测胶原纤维的面积百分比。

1.5 主要观察指标皮肤伤口的愈合情况。

1.6 统计学分析采用SPSS 13.0统计软件进行分析，数据以x(_)±s表示，组间比较采用单因素方差分析，两两比较采用LSD法。P < 0.05为差异有显著性意义。

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程